Adriamycin is a potent, broad-spectrum chemotherapeutic agent effective against solid
tumors and malignant
hematological disease. The major limiting factor for
adriamycin is its
cardiotoxicity. Thus, the objective of this study was to investigate the role of cardiomyocyte and endothelial cell apoptosis in
adriamycin-induced
cardiomyopathy, in vivo and in vitro. For in vivo study,
intraperitoneal injections of
adriamycin were administered to nine adult male Wistar rats and
normal saline to six rats as control. Eight of the nine rats in the
adriamycin group, but none in the control group, developed marked
ascites and
DNA ladders in
agarose gel electrophoresis of genomic
DNA extracted from the rat hearts (P<0.001). The ratio of apoptotic nuclei in the cardiomyocytes was significantly higher for the
adriamycin-treated rats (162+/-149/10(6) cells) than for the controls (4.2+/-1.3/10(6) cells; P<0.01) by TUNEL assay. Increased endothelial cell apoptosis was detected in the small coronary vessels of the myocardium of the
adriamycin-treated rats. Increased immuno-reactive
Caspase-3 expression was also noted for both cardiomyocytes and endothelial cells of
adriamycin-treated rats. In vitro
adriamycin treatment for cultured neonatal rat cardiomyocytes and human umbilical vein endothelial cells, respectively, showed a dose-related increase in apoptosis as determined by flowcytometry,
DNA ladder analysis, TUNEL assay and/or electron-microscope examination. A dose-related increase in the expression of
Fas antigen, Bax and
Caspase-3, as well as a decrease in the expression of Bcl-2, were determined for the
adriamycin-treated cardiomyocytes using Northern blot analysis,
reverse transcriptase polymerase chain reaction (RT-PCR) and
ribonuclease protection assay. RT-PCR also revealed increased
Fas antigen expression, decreased Bcl-2 expression, and no change in Bax expression for the
adriamycin-treated human umbilical vein cells. Further, pretreatment with broad
caspase inhibitor, but not neutralizing FasL antibody, resulted in inhibition of
adriamycin-induced endothelial cell apoptosis. In conclusion, these results indicate that both
adriamycin-induced cardiomyocyte and endothelial cell death can occur via apoptosis which is dose-related, and can occur both in vitro and in vivo with changes in the expression of the apoptosis-related genes.
Adriamycin-induced endothelial cell apoptosis is mediated by
caspase activation but is Fas/FasL signal pathway independent. Our data provides evidence that both cardiomyocyte and endothelial cell apoptosis may play an important role in
adriamycin-induced
cardiomyopathy.